In scanning probe microscopy, i.e. atomic force microscopy, as in other micromechnical cantilever applications, optical detection of the static deflection or the movements or oscillations of the cantilever has become a kind of standard for many applications. There are numerous devices using this kind of readout marketed by various manufacturers, one of them being a scanning probe microscope sold under the name “easyScan” by the assignee of this patent application.
Science has become interested in investigating materials in fluids, e.g. investigating biological samples like cells or fibers in various gaseous and/or liquid environments, using either scanning probe microscopes or other cantilever-based devices with an optical readout. This is usually accomplished by placing the sample and the scanning cantilever in a small vessel filled, at least to some extent, with the liquid or fluid in which the sample is to be investigated.
EP application 388 023 and U.S. Pat. No. RE 34,489 by P. K. Hansma et. al describe an AFM with a replaceable sample-carrying module which includes a provision for forming a fluid cell around the sample. The readout—or “positional sensing” as it is named in the subject patent/application—is shown as an optical system located outside the module. The module is factory set up, but must be fine tuned by the user. It is not mentioned which effect a change of the refractive index of the fluid within the module on the fine tuning has, but basic optical principles indicate that the fine tuning must also balance any change of the refractive index of the fluid surrounding the sample.
EP application 564 088 and U.S. Pat. No. 5,821,409 by K. Honma et al. describe a combined near field optical/AFM microscope whose sample is immersed in a liquid and both optically observed and cantilever-scanned. Though Honma et al. show quite a number of optical elements in their device, both for observation and scanning, nothing is said about the effect of the refractive indices of the fluids used.
U.S. Pat. No. 5,319,960 by Gamble et al. discloses an AFM capable of scanning a sample in contact with a fluid. However, the description of the optical detector system therein does not address any implications based on measuring in a liquid.
Similarly, U.S. Pat. No. 5,291,775 by Gamble et al. discloses another “scanning force microscope” with integrated optics, capable of measuring a sample in a fluid cell. Again, the fact that a fluid changes the properties of the optical path is nowhere addressed.
International application WO 98/10458 by P. K. Hansma et al. shows a further AFM with a complex optical readout system, generating a well-defined beam spot on the cantilever for measuring the deflection of the latter. Though it is described that the sample may be immersed in a fluid, e.g. water, the matter of a changed refractive index of the fluid used is not addressed.
EP Patent Specification 1 004 014 and U.S. Pat. No. 6,396,580 by Tewes et al. discloses an apparatus for fluorescence correlation spectroscopy, in particular for multi-color fluorescence correlation spectroscopy, in which apparatus light beams of different frequencies have to be focused in a transparent medium. To avoid any errors introduced by refractive optics, it provides a reflective optical system within the transparent medium for focussing the incident light beam. Though the disclosed system addresses some of the issues occurring when fluids having different refractive indices are changed within an optical beam path, the disclosed solution of a reflector within a probe chamber seems hardly adaptable to any of the typical cantilever-based SPMs. The reason is that in the latter the cantilever would intersect the incident light beam and the focusing would occur on the wrong side of the cantilever.
One specific characteristic is common to all prior art disclosures above: they use a planar interface in the optical path for entering or exiting the probe chamber.
In a different technology, i.e. the technology of optical immersion microscopes, lenses with a concave surface facing an immersion liquid are known. However, as shown in U.S. Pat. No. 5,517,360 by Suzuki and U.S. Pat. No. 5,805,346 by Tomimatso, both addressing immersion microscope objectives, the disclosed objectives are extremely complex designs, consisting of ten or more lenses with detailed specifications. The issue of changing refractive indices of the immersion fluid is not addressed. The lenses closest to the object are shown as “positive” meniscus lenses, i.e. converging lenses, having a concave surface facing this object and contacting the liquid. The radius of this surface determined by the refractive indices of the various materials used for the many parts of the objective—it is not related to and differs from the distance to the object or to a desired focal point. Contrary to the above, as will be shown and explained below, the meniscus lens according to one embodiment of the present invention is a “neutral” lens, the radius of its inner surface being dependent on the distance to a desired focal point.
The same is true, mutatis mutandis, for the immersion microscope objective disclosed in U.S. Pat. No. 7,262,922 by Yamaguchi. In this patent, the lens closest to the object is described as exhibiting a positive refraction and having a radius of greater than 1 up to 50 times the focal length of the objective. Both conditions or measures differ from the rules give for the “neutral” meniscus lens according to one embodiment of the present invention.
Reverting back to SPM and the like, the point is that whenever an optical system outside the fluid-filled probe chamber or just outside the fluid is used and the fluid's refractive index changes, the optical paths change between the light source and the cantilever as well as between the cantilever and the optical receiver. This usually needs a re-adjustment of the optical system.
It seems that the issue of re-adjustment and/or re-calibration was not recognized or it was considered immaterial or not sufficiently important in a scientific environment.
There are two significant points to understand in this respect whenever the refractive index of the fluid within a probe chamber changes.
(1) Assumed that an incident beam, typically a cone, enters the probe chamber perpendicularly, usually through a window of a transparent material, and is focused correctly onto a cantilever in a first fluid. Now, when the first fluid is replaced by a second fluid with a different refractive index, the beam path remains essentially unaffected, but not the beam's focus on the cantilever, better: the beam will not focus any more on the same spot (in the same plane) as previously. Depending on the overall design of the detection system, the same may be true, mutatis mutandis, for the reflected beam when it leaves the chamber perpendicularly, passing the chamber wall again through a window of a transparent material: its focal plane shifts whenever the refractive index of the fluid within the chamber changes. In this case, the detector receiving the reflected beam will in most cases provide a different output.
(2) It becomes worse when the incident beam enters the probe chamber under an angle or the reflected beam leaves the probe chamber under an angle, i.e. passes the chamber walls not perpendicularly. In this case, both the incident and the reflected beams will follow another path a soon as the refractive index of the fluid within the chamber changes. Thus the incident beam may not be lined up with the cantilever any more or the reflected beam may not hit the detector—or both.
It seems that until now, re-adjustment and/or re-calibration were considered the only solutions to this problem in scanning probe microscopy. Though this may be acceptable in a test or laboratory environment, it is certainly not acceptable in an industrial or manufacturing environment. Also, it makes measurements whereby the fluid is to be changed under way, i.e. during the measurement, practically impossible.